Method for Freeze-Drying Nucleic Acid/Block Copolymer/Cationic Surfactant Complexes

ABSTRACT

This invention relates generally to the freeze-drying of formulations comprising a polynucleotide, a block copolymer and a cationic surfactant. In the presence of a cryoprotectant or bulking agent, a formulation can be freeze-dried, whereby upon reconstitution of the dried formulation, the microparticles maintain their optimal size and aggregation or fusion is avoided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/725,009 filed Dec. 2, 2003, now pending; which claims the benefitunder 35 USC § 119(e) to U.S. Application Ser. No. 60/435,273 filed Dec.23, 2002, now abandoned. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the freeze-drying of formulationscomprising a polynucleotide, a block copolymer and a cationicsurfactant. In the presence of a cryoprotectant or bulking agent, aformulation can be freeze-dried, whereby upon reconstitution of thedried formulation, the microparticles maintain their optimal size andaggregation or fusion is avoided.

2. Related Art

The use of non-ionic block copolymers as adjuvants in polynucleotidebased medicaments has been documented in the art. Polynucleotidecomplexes which comprise a polynucleotide, a block copolymer and acationic surfactant have demonstrated enhanced in vivo immune response.In some cases, it is desirable or necessary to supply a suspension ofthese complexes in a dry powder form that can be reconstituted to anaqueous system just prior to use. One method of drying an aqueous mediumis by lyophilization in which the medium is frozen and then the water isextracted by sublimation under vacuum. If the aqueous medium contains asuspension of microparticles, these microparticles tend to clusterduring the initial freezing step of the lyophilization process due tothe propagation of the crystallization front. Often, the microparticlesbecome permanently aggregated and do not redisperse when reconstituted,creating a population with a very polydisperse size distribution.

Methods for controlling aggregation during freeze-drying andreconstitution through the use of cryoprotectants and bulking agents orother excipients are known in the art and have been described forliposome formulations (See U.S. Pat. No. 5,817,334, hereby incorporatedby reference in its entirety), microparticles (See U.S. Pat. No.6,482,581, hereby incorporated by reference in its entirety) and nucleicacid-polycation compositions (See U.S. Pat. No. 6,251,599, herebyincorporated by reference in its entirety).

Despite these advances, there exists a need for methods by which tofreeze-dry and reconstitute compositions comprising a polynucleotide anda block copolymer that also contain a cationic surfactant, such that thesame particle size and population polydispersity prior to freeze-dryingare maintained following reconstitution. The present invention fulfillsthis need.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of preparing alyophilized composition comprising mixing a block copolymer with apopulation of polynucleotide molecules, a cationic surfactant, and anamorphous cryoprotectant or a bulking agent or any combination thereof,at a temperature below the cloud point of the block copolymer to form amixture. This mixture is then frozen and finally dried under vacuum.Upon reconstitution of this mixture, the particle size and populationpolydispersity are maintained.

In a further embodiment, the composition comprising a polynucleotide, ablock copolymer and a cationic surfactant are mixed at a temperaturebelow the cloud point of the block copolymer, at a temperature of about−2° C. to about 8° C. In yet another aspect of the invention, prior tolyophilization, the mixture is cold filtered, thereby rendering itsterile. Suitably, this cold filtration step is performed at atemperature of about −2° C. to about 8° C. using a filter with a poresize of about 0.01 microns to about 2 microns.

Block copolymers such as polyoxyethylene (POE)/polyoxypropylene (POP)are desired. An example of a useful block copolymer is PoloxamerCRL-1005. Suitable cationic surfactants for use in the present inventioninclude benzalkonium chloride and(±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminiumchloride (Pr-DoctRIe-OAc).

Suitable amorphous cryoprotectants and crystalline bulking agentsinclude the following sugars: sucrose, lactose, trehalose, maltose orglucose, wherein the solution comprises about 1% to about 20% (w/v)sugar. A suitable embodiment of the invention contains about 10%sucrose. The composition may also optionally comprise a pH stabilizingbuffer.

The present invention also encompasses the polynucleotide/blockcopolymer/cationic surfactant microparticles produced by thelyophilization processes described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are graphs plotting the Z average mean particle sizeand polydispersity of reconstituted lyophilized microparticles producedby the method described in Example 1.

FIG. 2 contains the structures of the following cationic lipids:Benzalkonium chloride (BAK C12),(±)-N-(Benzyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanaminium bromide(Bn-DHxRIE),(±)-N-(2-Acetoxyethyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanaminiumbromide (DHxRIE-OAc),(±)-N-(2-Benzoyloxyethyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanraminiumbromide (DHxRIE-OBz) and(±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminiumchloride (Pr-DOctRIE-OAc).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “PBS” refers to—phosphate buffered saline—.

As used herein, “BAK” refers to—benzalkonium chloride—.

As used herein, “BEC” refers to—benzethonium chloride—.

As used herein, “CPC” refers to—cetylpyridinium chloride—.

As used herein, “CTAC” refers to—cetyl trimethyl-ammonium chloride—.

As used herein, “PS-80” refers to—polysorbate 80—.

As used herein, the term “cloud point” refers to the point in atemperature shift, or other titration, at which a clear solution becomescloudy, i.e., when a component dissolved in a solution begins toprecipitate out of solution.

As used herein, the words “particle” and “microparticle” areinterchangeable.

As used herein, the words “mixture” and “solution” are interchangeable.

As used herein, the term “adjuvant” is any substance or combination ofsubstances which nonspecifically enhances the immune response to anantigen; and also relates to any substance which enhances the immuneresponse directly related to delivery of a polynucleotide within avertebrate or mammalian host, such as a human or non-human mammalianhost, such that administration of the adjuvant in combination with thepolynucleotide results in an increased in vivo immune response toexpression of the intended antigen or antigens encoded by thepolynucleotide. Included in this definition are substances which may actas facilitators of gene delivery, thereby increasing the amount ofplasmid DNA delivered to cells that can express the intended antigen.Substances which may enhance delivery of plasmid DNA would include thosewhich do not substantially interact with the plasmid DNA in theformulation and substances which do interact with the plasmid DNA,forming tightly bound or weakly bound complexes between the adjuvant andthe plasmid DNA, either in vitro or in vivo.

As used herein, the term “polynucleotide” is a nucleic acid moleculewhich contains essential regulatory elements such that upon introductioninto a living, vertebrate cell, the nucleic acid molecule is able todirect the cellular machinery to produce translation products encoded bythe genes comprising the nucleic acid molecule.

As used herein, the term “polynucleotide medicament” is used to indicatepolynucleotide-based compositions, including compositions which comprisethe poloxamers and cationic surfactants disclosed herein, useful as avehicle to deliver a transgene of interest to a vertebrate host, such asa human or non-human mammalian host, or to provide or promote detectableand/or therapeutic levels of expression of the transgene, and/or togenerate or promote an immune response to the expression product of thetransgene.

As used herein, the term “vector” refers to a vehicle by which DNAfragments, most likely comprising a transgene or portion thereof whichexpresses an antigen or antigenic epitope, can be introduced into a hostorganism or host tissue. There are various types of vectors whichinclude but are not limited to recombinant vectors, including DNAplasmid vectors, recombinant viral vectors such as adenovirus vectors,retrovirus vectors and adeno-associated virus vectors, as well asbacteriophage vectors and cosmid vectors.

The term “gene” or “transgene” refers to a segment of a nucleic acidmolecule which encodes a discrete protein or a portion thereof, such asa portion of the full-length protein which will induce an appropriateimmune response within the host.

As used herein, the term “amorphous cryoprotectant” refers to a compoundwhich, when included in the formulations of the present invention duringfreezing or lyophilization under given conditions, does not formcrystals. It is specifically intended that compounds that are known toform crystals under certain lyophilization conditions but not underothers are included within the term “amorphous cryoprotectant,” so longas they remain amorphous under the specific freezing or lyophilizationconditions to which they are subjected. The term “cryoprotectant” may beused interchangeably with the term “amorphous cryoprotectant” herein.

As used herein, “crystalline bulking agent” refers to a compound which,when included in the formulations of the present invention duringfreezing or lyophilization under given conditions, forms crystals. It isspecifically intended that compounds that are known to form crystalsunder certain lyophilization conditions but not under others areincluded within the term “crystalline bulking agent,” so long as theycrystallize under the specific freezing or lyophilization conditions towhich they are subjected. The term “bulking agent” may be usedinterchangeably with the term “crystalline bulking agent” herein.

As used herein, “lyophilization” is a means of drying, achieved byfreezing a wet substance at a temperature from about −172° C. to about−2° C. followed by rapid dehydration by sublimation under a vacuum leveldown to the lower level of a diffusion pump. A useful pressure range isfrom about 0.1 mTorr to about 0.5 Torr. The term “freeze-drying” may beused interchangeably with the term “lyophilization” herein.

Amorphous cryoprotectants, crystalline bulking agents, and methods ofdetermining the same are known and available in the art. The followingarticles, incorporated herein by reference, provide a basic teaching inthis regard: Osterberg et al., Pharm Res 14(7):892-898 (1997); Oliyai etal., Pharm Res 11(6):901-908 (1994); Corveleyn et al., Pharm Res13(1):146-150 (1996); Kim et al., J. Pharm Sciences 87(8):931-935(1998); Martini et al., PDA J. Pharm Sci Tech 51(2):62-67 (1997);Martini et al., STP Pharma Sci. 7(5):377-381 (1997); and Orizio et al.,Boll. Chim. Farm. 132(9):368-374 (1993).

The present invention relates to novel methods for lyophilizing aformulation suitable for use in polynucleotide based medicaments. Themethods result in formulations comprising polynucleotide molecules,block copolymer, and a cationic surfactant that upon reconstitutionmaintain the same particle size and polydispersity prior tofreeze-drying.

The method of the present invention comprises mixing: (i) a cationicsurfactant; (ii) a block copolymer; and (iii) a polynucleotide; at atemperature below the cloud point of said block copolymer to form amixture. The order in which components of the mixture are added mayvary. A suitable order in which ingredients of the mixture may be addedinclude, but is not limited to, (i) polynucleotide; (ii) blockcopolymer; and (iii) cationic surfactant. Alternatively, the order ofaddition can also include: (i) cationic surfactant; (ii) blockcopolymer; and (iii) polynucleotide. Stirring of the mixture can occuronce all components have been added, concurrently while components arebeing added, or in between the addition of components.

The block copolymers useful in the polynucleotide based medicamentsdescribed herein are block copolymers which form microparticles at roomtemperature (above the block copolymer cloud point) and may associatewith a population of nucleic acid molecules, such as a population ofplasmid DNA molecules, with and without the addition of cationicsurfactants. The nucleic acid molecules of the present invention mayinclude a deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA) as well as a ribonucleic acid molecule (RNA).In regard to the block copolymer, a suitable group of copolymers used inthe methods of the present invention include non-ionic block copolymerswhich comprise blocks of polyoxyethylene (POE) and polyoxypropylene(POP).

While the invention contemplates the use of any such block copolymerwhich promotes generation of a particle size and surface charge asdescribed herein, suitable non-ionic block copolymers includepolyoxyethylene (POE)/polyoxypropylene (POP) block copolymers,especially higher molecular weight POE-POP-POE block copolymers. Thesecompounds are described in U.S. Reissue Pat. No. 36,665, U.S. Pat. No.5,567,859, U.S. Pat. No. 5,691,387, U.S. Pat. No. 5,696,298 and U.S.Pat. No. 5,990,241, and Published International Patent Application WO96/04392, all of which are hereby incorporated by reference.

Briefly, these non-ionic block copolymers have the following generalformula: HO(C₂H₄O)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)H wherein (y) represents anumber such that the molecular weight of the hydrophobic POP portion(C₃H₆O) is up to approximately 20,000 daltons and wherein (x) representsa number such that the percentage of hydrophilic POE portion (C₂H₄O) isbetween approximately 1% and 50% by weight.

A suitable POE-POP-POE block copolymer that can be used in the inventionhas the following formula HO(C₂H₄O)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)H wherein(y) represents a number such that the molecular Weight of the hydrophobe(C₃H₆O) is between approximately 9,000 Daltons and 15,000 Daltons and(x) represents a number such that the percentage of hydrophile (C₂H₄O)is between approximately 3% and 35%.

Another suitable POE-POP-POE block copolymer that can be used in theinvention has the following formula: HO(C₂HO)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)Hwherein (y) represents a number such that the molecular weight of thehydrophobe (C₃H₆O) is between approximately 9,000 Daltons and 15,000Daltons and (x) represents a number such that the percentage ofhydrophile (C₂H₄O) is between approximately 3% and 10%.

A typical POE/POP block copolymer utilized herein will comprise thestructure of POE-POP-POE, as reviewed in Newman et al. Critical Reviewsin Therapeutic Drug Carrier Systems 15 (2): 89-142 (1998). A suitableblock copolymer for use in the methods of the present invention is aPOE-POP-POE block copolymer with a central POP block having a molecularweight in a range from under 1000 daltons up to approximately 20,000daltons and flanking POE blocks which comprise up to about 50% of thetotal molecular weight of the copolymer. Block copolymers such as these,which are much larger than earlier disclosed Pluronic-based POE/POPblock copolymers, are described in detail in U.S. Reissue Pat. No.36,655. A representative POE-POP-POE block copolymer utilized toexemplify DNA formulations of the present invention is disclosed inPublished International Patent Application No. WO 96/04392, is alsodescribed at length in Newman et al. (Id.), and is referred to asCRL-1005 (CytRx Corp).

CRL-1005 is another suitable surface-active copolymer that can be usedin the invention and has the following formula:HO(C₂H₄O)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 12,000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 5%. In the case ofCRL-1005, (x) is about 7, ±1 and (y) is approximately 12,000 Daltons,with about 207 units, ±7.

Although there is evidence to suggest that the association of plasmidDNA to the CRL-1005 particles leads to an improved immune response, themechanism by which the immune response is enhanced is at presentunclear. While not being bound by theory in any way, it is possible thatDNA associated to CRL-1005 particles may be more readily taken up andexpressed by cells. It is also possible that the negative surface chargeof the CRL-1005 particles, produced by the association of plasmid DNA toCRL-1005/BAK particles, may be important for enhancing the adjuvantproperties of CRL-1005. The current invention does not distinguishbetween these two possible mechanisms of enhancing the immune response.

The measurement of surface charge (zeta potential) and the amount of DNAassociated with CRL-1005 particles are consistent with a model for theinteraction of plasmid DNA/the block copolymer (CRL-1005) and thecationic surfactant (for example, BAK). The model suggests that BAKassociating with particles of CRL-1005, through hydrophobicinteractions, results in a reduction of the CRL-1005 particle size andin the formation of positively charged CRL-1005 particles. Associationof the polynucleotide (plasmid DNA) is believed to occur throughelectrostatic interactions between the positively charged headgroup ofthe cationic surfactant (BAK) and the DNA phosphate groups, while thehydrophobic tail of the cationic surfactant is embedded within the blockcopolymer (CRL-1005) particle.

Published International Patent Application WO 02/00844 discloses thatthe generation of physically distinct particles comprising the blockco-polymer CRL-1005, a cationic surfactant and DNA, further promotes theassociation of plasmid DNA to the block copolymer as compared to theblock co-polymer and DNA alone. The particles containing all threecomponents also resulted in a marked enhancement of a cellular immuneresponse.

An alternative surface-active copolymer that can be used in theinvention has the following formula:HO(C₂H₄O)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 9,000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 3-5%.

Another suitable surface-active copolymer that can be used in theinvention has the following formula:HO(C₂H₄O)_(x)(C₃H₆O)_(y)(C₂H₄O)_(x)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 9,000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 3%.

In yet another alternative embodiment the present invention relates to amethod for producing a lyophilized formulation suitable for use inpolynucleotide based medicaments comprising a block copolymer which isPoloxamer CRL-1005.

Another suitable group of block copolymers for use in the presentinvention include “reverse” block copolymers wherein the hydrophobicportions of the molecule (C₃H₆O) and the hydrophilic portions (C₂H₄O)have been reversed such that the polymer has the formula:HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobic POP portion (C₃H₆O) isup to approximately 20,000 daltons and wherein (x) represents a numbersuch that the percentage of hydrophilic POE portion (C₂H₄O) is betweenapproximately 1% and 50% by weight. These “reverse” block copolymershave the structure POP-POE-POP and are described in U.S. Pat. Nos.5,656,611 and 6,359,054.

A suitable POP-POE-POP block copolymer that can be used in the inventionhas the following formula: HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein(y) represents a number such that the molecular weight of the hydrophobe(C₃H₆O) is between approximately 9,000 Daltons and 15,000 Daltons and(x) represents a number such that the percentage of hydrophile (C₂H₄O)is between approximately 3% and 35%.

Another suitable POP-POE-POP block copolymer that can be used in theinvention has the following formula:HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) is betweenapproximately 9,000 Daltons and 15,000 Daltons and (x) represents anumber such that the percentage of hydrophile (C₂H₄O) is betweenapproximately 3% and 10%.

Another suitable surface-active copolymer that can be used in theinvention and has the following formula:HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 12,000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 5%.

An alternative surface-active copolymer that can be used in theinvention has the following formula:HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 9,000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 3-5%.

Another suitable surface-active copolymer that can be used in theinvention has the following formula:HO(C₃H₆O)_(y)(C₂H₄O)_(x)(C₃H₆O)_(y)H wherein (y) represents a numbersuch that the molecular weight of the hydrophobe (C₃H₆O) isapproximately 9000 Daltons and (x) represents a number such that thepercentage of hydrophile (C₂H₄O) is approximately 3%.

The block copolymers for use in the invention are amphipathic compoundswith inverse solubility characteristics in aqueous media. Below theircloud points (1-20° C.), these copolymers are water-soluble and formclear solutions that can be filter sterilized. The solution processinvolves the formation of hydrogen bonds between oxygen atoms andhydroxyl groups in the copolymer and water molecules. When a solution ofcopolymer is warmed and passes through its cloud point, the increasedthermal motion is sufficient to break the hydrogen bonds and as thecopolymer comes out of solution, they self-assemble into microparticles(See Todd, C. W., et al. Vaccine 15. 564-570 (1997) and Todd, C. W., etal. Dev. Biol. Stand. 92: 343-353 (1997)). The process is reversible.

Any type of polynucleotide can be incorporated into the method of thecurrent invention. For example plasmid DNA, genomic DNA, cDNA, DNAfragments and RNA. Certain formulations of the present invention includea cocktail of plasmids. Various plasmids desired in a cocktail arecombined together in PBS or other diluent prior to addition to the otheringredients. There is no upper limit to the number of different types ofplasmids which can be used in the methods of the present invention.Furthermore, plasmids may be present in a cocktail at equal proportions,or the ratios may be adjusted based on, for example, relative expressionlevels of the antigens or the relative immunogenicity of the encodedantigens. Thus, various plasmids in the cocktail may be present in equalproportion, or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times as much of oneplasmid may be included relative to other plasmids in the cocktail.

The polynucleotide formulations produced by the methods of the presentinvention also comprise a cationic surfactant. It will be known to oneof skill in the art that numerous cationic surfactants may be acandidate for use in these formulations. Therefore, the inventioncontemplates use of any cationic surfactant which, along with a blockcopolymer, and a polynucleotide promotes generation of a particle sizeand surface charge as described herein. Cationic surfactants which maybe used include, but are not limited to, benzalkonium chloride (BAK),benzethonium chloride, cetramide (which containstetradecyltrimethylammonium bromide and possibly small amounts ofdedecyltrimethylammonium bromide and hexadecyltrimethyl ammoniumbromide), cetylpyridinium chloride (CPC) and cetyl trimethylammoniumchloride (CTAC), primary amines, secondary amines, tertiary amines,including but not limited to N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, other quaternary amine salts,including but not limited to dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemethanaminium chloride (DEBDA), dialkyldimethylammoniumsalts, -[1-(2,3-dioleyloxy)-propyl]-N,N,N, trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl dioleoyl), 1,2-diacyl-3 (dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio) butanoyl-sn-glycerol,1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl(4′-trimethylammonio) butanoate), N-alkyl pyridinium salts (e.g.cetylpyridinium bromide and cetylpyridinium chloride),N-alkylpiperidinium salts, dicationic bolaform electrolytes (C₁₂Me₆;C₁₂Bu₆), dialkylglycetylphosphorylcholine, lysolecithin, L-adioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester,lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhCnN⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluCnN⁺), cationic derivatives of cholesterol,including but not limited tocholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt,cholesteryl-3β-oxysuccinamidoethylenedimethylamine,cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt,cholesteryl-3β-carboxyamidoethylenedimethylamine, and3β-[N-(N′,N′-dimethylaminoetanecarbomoyl) cholesterol] (DC-Chol).

Other examples of cationic surfactants for use in the invention areselected from the group of cationic lipids includingN-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammoniumbromide (PA-DEMO),N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammoniumbromide (PA-DELO),N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide(PA-TELO), andN¹-(3-aminopropyl)((2-dodecyloxy)ethyl)-N²-(2-dodecyloxy)ethyl-1-piperazinaminiumbromide (GA-LOE-BP),DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORIdiester), and1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORIester/ether).

Additional suitable, but non-limiting cationic lipids for use in certainembodiments of the present invention include DMRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide), GAP-DMORIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide), and GAP-DLRIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanaminiumbromide).

Other cationic lipids for use in the present invention include thecompounds described in U.S. Pat. Nos. 5,264,618, 5,459,127 and5,994,317. Non-limiting examples of these cationic lipids include(±)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)-1-propaniminium pentahydrochloride (DOSPA),(±)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminiumbromide (β-aminoethyl-DMRIE or βAE-DMRIE) and(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide (GAP-DLRIE).

Other examples of DMRIE-derived cationic lipids that are useful in thepresent invention include(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminiumbromide (GAP-DDRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanaminiumbromide (GAP-DMRIE),(±)-N-((N″-methyl)-N′-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (GMU-DMRIE),(±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (DLRIE), and(±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)propyl-1-propaniminiumbromide (HP-DORIE).

In a suitable aspect of the present invention, the cationic surfactantis selected from the group consisting of benzalkonium chloride,benzethonium chloride, cetramide, cetylpyridinium chloride and cetyltrimethylammonium-chloride. Benzalkonium chloride is availablecommercially and is known to exist as a mixture ofalkylbenzyldimethylammonium chlorides of the general formula:[C₆H₅CH₂N(CH₃)2R] C1, where R represents a mixture of alkyls, includingall or some of the group beginning with n-C₈H₁₇ through n-C₁₆H₃₃. Theaverage MW of BAK is 360 (see Handbook of Pharmaceutical Excipients, Ed.Wade and Weller, 1994, 2nd Ed. at page 27-29). Benzethonium chloride isN,N-dimethyl-N-[2-[2-[4-(1,1,3,3tetramethylbutyl)phenoxy]ethoxy]ethyl]benzene-methanaminiumchloride (C₂₇H₄₂CINO₂), which has a molecular weight of 448.10 (Handbookof Pharmaceutical Excipients at page 30-31). Cetramide consists mainlyof trimethyltetradecylammonium bromide (C₁₇H₃₈BrN), which may containsmaller amounts of dodecyltrimethyl-ammonium bromide (C₁₅H₃₄BrN) andhexadecyltrimethylammonium bromide (C₁₉H₄₂BrN), and has a molecularweight of 336.40 (Handbook of Pharmaceutical Excipients at page 96-98).

In another suitable aspect of the present invention, the cationicsurfactant for use in the methods of the current invention is selectedfrom the group consisting of(±)-N-(Benzyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanaminium bromide(Bn-DHxRIE),(±)-N-(2-Acetoxyethyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanaminiumbromide (DHxRIE-OAc),(±)-N-(2-Benzoyloxyethyl)-N,N-dimethyl-2,3-bis(hexyloxy)-1-propanaminiumbromide (DHxRIE-OBz) and(±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminiumchloride (Pr-DOctRIE-OAc). These lipids are disclosed in U.S.Provisional Application No. 60/435,303. In another suitable aspect ofthe present invention, the cationic surfactant is Pr-DOctRIE-OAc.

Auxiliary agents for use in compositions of the present inventioninclude, but are not limited to, non-ionic detergents and surfactantsIGEPAL CA 630® CA 630, NONIDET NP-40, Nonidet® P40, Tween-20®,Tween-80®, Triton X-100™, and Triton X-114™; the anionic detergentsodium dodecyl sulfate (SDS); the sugar stachyose; the condensing agentDMSO; and the chelator/DNAse inhibitor EDTA. In certain specificembodiments, the auxiliary agent is DMSO, Nonidet P40®. See, e.g., U.S.Patent Application Publication 20020019358, published Feb. 14, 2002,which is incorporated herein by reference in its entirety.

The polynucleotide formulations produced by the methods of the presentinvention may also optionally include a non-ionic surfactant, such aspolysorbate-80, which may be a useful excipient to control particleaggregation in the presence of the polynucleotide. Additional non-ionicsurfactants are known in the art and may be used to practice thisportion of the invention. These additional non-ionic surfactantsinclude, but are not limited to, other polysorbates, -Alkylphenylpolyoxyethylene ether, n-alkyl polyoxyethylene ethers (e.g., Tritons™),sorbitan esters (e.g., Spans™), polyglycol ether surfactants(Tergitol™), polyoxyethylenesorbitan (e.g., Tweens™), poly-oxyethylatedglycol monoethers (e.g., Brij™, polyoxylethylene 9 lauryl ether,polyoxyethylene 10 ether, polyoxylethylene 10 tridecyl ether), lubrol,perfluoroalkyl polyoxylated amides,N,N-bis[3D-gluconamidopropyl]cholamide, decanoyl-N-methylglucamide,-decyl β-D-glucopyranozide, n-decyl β-D-glucopyranozide, n-decylβ-D-maltopyanozide, ndodecyl β-D-glucopyranozide, n-undecylβ-D-glucopyranozide, n-heptyl β-D-glucopyranozide, n-heptylβ-D-thioglucopyranozide, n-hexyl β-D-glucopyranozide, n-nonanoylβ-glucopyranozide 1-monooleyl-rac-glycerol, nonanoyl-N-methylglucamide,-dodecyl β-D-maltoside, N,N bis[3-gluconamidepropyl]deoxycholamide,diethylene glycol monopentyl ether, digitonin,hepanoyl-N-methylglucamide, octanoyl-N-methylglucamide, n-octylβ-D-glucopyranozide, n-octyl β-D-glucopyranozide, n-octylβ-D-thiogalactopyranozide, n-octyl β-D-thioglucopyranozide.

The present invention relates to methods for lyophilizing a formulationsuitable for use in polynucleotide based medicaments such that uponreconstitution, the particle size and population polydispersity of themicroparticles remain unchanged. The methods result in the generation ofmicroparticles (at temperatures above the cloud point of CRL-1005, oranother representative block copolymer) which comprise a block copolymerand cationic surfactant in contact with polynucleotide molecules. Thecomponents which will eventually comprise the microparticles are mixedwith an amorphous cryoprotectant or a crystalline bulking agent bystirring at temperature below the cloud point of the block copolymer.Additionally, the mixture may also comprise a pH stabilizing buffer.Each component of this mixture must be thoroughly co-dissolved at atemperature below the cloud point of the polymer. This solution can alsobe sterilized via cold filtration and aliquoted into sterile vials priorto lyophilization. Prior to administration to a patient by injection, orany other means, the freeze-dried formulation can be reconstituted andthe vial can be brought to room temperature or to a temperature abovethe cloud point of the block copolymer, wherein microparticle formationwill occur during the warming process. It is the discovery of thecurrent inventors that microparticle reconstitution results in particleswith a particle size and a population polydispersity that remainsunchanged during the freeze-drying process. This is further illustratedin Examples 1 and 2. Table 1 indicates the mean average diameter ofmicroparticles composed of polynucleotide, poloxamer CRL-1005 and BAKco-dissolved in solutions of both PBS and 10% sucrose/10 mM sodiumphosphate. The microparticles prepared in PBS have a much largerpolydispersity following reconstitution than prior to freeze-dryingindicating a broad size range of particles. In contrast, themicroparticles co-dissolved in 10% sucrose/10 mM sodium phosphatedemonstrate virtually the same polydispersity before lyophilization andafter reconstitution, indicating a narrow size distribution. FIGS. 1Aand 1B demonstrate that particle size and polydispersity remainunchanged after 6 hours at room temperature following reconstitution.

The lyophilized composition of the present invention may bereconstituted in any aqueous solution which produces a stable,mono-dispersed solution suitable for administration. Such aqueoussolutions include, but are not limited to: sterile water, PBS or saline.

Upon review of this specification, the artisan will be able to mix andmatch various block copolymers, cationic surfactants, excipients, aswell as utilize various concentrations of these components. The artisanwill be able to measure in vitro structural characteristics of theformulation, as shown herein, which may be instructive in preparing suchcomponents for in vivo administration.

In suitable embodiments of the present invention a polynucleotide ismixed with the poloxamer CRL-1005, BAK (Benzalkonium chloride 50%solution, available from Ruger Chemical Co. Inc.) and an amorphouscryoprotectant or crystalline bulking agent. Additionally, the mixturemay also comprise a pH stabilizing buffer. Suitable final concentrationsof each component of the formulae are described in the examples, but forany of these methods, the concentrations of each component may be variedby basic stoichiometric calculations known by those of ordinary skill inthe art to make a final solution having the desired concentrations.

In the method of the current invention, the concentration of the blockcopolymer is adjusted depending on, for example, transfectionefficiency, expression efficiency, or immunogenicity. In suitableembodiments, the final concentration of the block copolymer is betweenabout 1 mg/mL to about 75 mg/mL. Alternatively the final concentrationof the block copolymer is between about 3 mg/mL to about 50 mg/mL, about5 mg/mL to about 40 mg/mL, about 6 mg/mL to about 30 mg/mL. For example,about 6 mg/mL, about 6.5 mg/mL about 7 mg/mL, about 7.5 mg/mL, about 8mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL,about 25 mg/mL, or about 30 mg/mL of the block copolymer.

In another suitable embodiment of the method of the current invention,the concentration of the poloxamer CRL-1005 is adjusted depending on,for example, transfection efficiency, expression efficiency, orimmunogenicity. In suitable embodiments, the final concentration of thepoloxamer CRL-1005 is between about 1 mg/mL to about 75 mg/mL.Alternatively the final concentration of the poloxamer CRL-1005 isbetween about 3 mg/mL to about 50 mg/mL, about 5 mg/mL to about 40mg/mL, about 6 mg/mL to about 30 mg/mL. For example, about 6 mg/mL,about 6.5 mg/mL about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 9mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL,or about 30 mg/mL of CRL-1005. Similarly the concentration of DNA in themethods of the current invention is adjusted depending on many factors,including the amount of a formulation to be delivered, the age andweight of the subject, the delivery method and route and theimmunogenicity of the antigen being delivered. In an alternativeembodiment, the final concentration of DNA is from about 1 mg/mL toabout 30 mg/mL of plasmid (or other polynucleotide). Alternatively, aformulation of the present invention may have a final concentration ofDNA from about 0.1 mg/mL to about 20 mg/mL, or about 1 mg/mL to about 10mg/mL. For example, the final DNA concentration may be about 1 mg/mL,about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL,about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, or about 20mg/mL.

Additionally, the concentration of the cationic surfactant may beadjusted depending on, for example, a desired particle size and improvedstability. Indeed, in certain embodiments, the methods of the presentinvention include CRL-1005 and DNA, but are free of cationic surfactant.In general, cationic surfactant-containing formulations of the presentinvention are adjusted to have a final concentration of cationicsurfactant from about 0.01 mM to about 5 mM. Suitably, a formulation ofthe present invention may have a final cationic surfactant concentrationof about 0.06 mM to about 1.2 mM, or about 0.1 mM to about 1 mM, orabout 0.2 mM to about 0.7 mM. For example, a formulation of the presentinvention may have a final cationic surfactant concentration of about0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, or about0.7 mM.

Additionally, the concentration of BAK may be adjusted depending on, forexample, a desired particle size and improved stability. Indeed, incertain embodiments, the methods of the present invention includeCRL-1005 and DNA, but are free of BAK. In general BAK-containingformulations of the present invention are adjusted to have a finalconcentration of BAK from about 0.01 mM to about 5 mM. Alternatively, aformulation of the present invention may have a final BAK concentrationof about 0.06 mM to about 1.2 mM, or about 0.1 mM to about 1 mM, orabout 0.2 mM to about 0.7 mM. For example, a formulation of the presentinvention may have a final BAK concentration of about 0.2 mM, about 0.3mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, or about 0.7 mM.

The total volume of the formulations produced by the methods of thecurrent invention may be scaled up or down, by choosing apparatus ofproportional size. Finally, in carrying out any of the methods describedbelow, the three components of the formulation, BAK, CRL-1005, andplasmid DNA, may be added in any order.

The polynucleotide based medicaments produced by the methods of thepresent invention may be formulated in any pharmaceutically effectiveformulation for host administration. It will be useful to utilizepharmaceutically acceptable formulations which also provide long-termstability of the polynucleotide based medicaments of the presentinvention. During storage as a pharmaceutical entity, DNA plasmidsundergo a physiochemical change in which the supercoiled plasmidconverts to the open circular and linear form. A variety of storageconditions (low pH, high temperature, low ionic strength) can acceleratethis process. Therefore, the removal and/or chelation of trace metalions (with succinic or malic acid, or with chelators containing multiplephosphate ligands, or with chelating agents such as EDTA) from the DNAplasmid solution, from the formulation buffers or from the vials andclosures, stabilizes the DNA plasmid from this degradation pathwayduring storage.

In one embodiment of the invention, the particles formed by the currentmethod are from about 100 nm to about 2000 nm in diameter. The non-ionicblock copolymer particle in the presence of the cationic surfactant willhave a positive surface charge whereas the polymer particle in thepresence of cationic surfactant and DNA should have a surface chargesignificantly more negative than the polymer particle alone. Theexemplified microparticles described in the Example sections range fromabout 200-600 nm in diameter with a slightly positive zeta potentialmeasurement in the presence of BAK but without addition of thepolynucleotide (about 2.5 mV for CRL-1005 and 0.71 mM BAK) and anegative zeta potential when the polynucleotide (at 5 mg/mL) is present(about −46.6 mV for CRL-1005 and 0.71 mM BAK and 5 mg/mL plasmid DNA).While these values are instructive, they are by no way limiting.

The addition of a cationic surfactant changes the configuration orstructural integrity of the particle, which in turn increases theability of the altered structure to better interact with polynucleotidemolecules. Therefore, while ranges of surface charge and sizemeasurements of various particles may be instructive, they are notnecessarily limiting. One of ordinary skill in the art can adjustconcentrations of one type of block copolymer and one type of cationicsurfactant to form distinct microparticles, wherein the microparticlesare ultimately characterized by an increased ability to associate with aspecific population of polynucleotide molecules.

The formulation produced by the methods of the current invention may bealiquoted into a suitable container for storage. Suitable containersinclude, but are not limited to, glass vials, glass bottles, syringes,sterilizable plastic bags, polyethylene tubes, vials or bottles, andpolypropylene tubes, vials or bottles and any other container suitablefor manufacturer bulk use or in the preparation of a kit comprising thepolynucleotide based medicaments of the invention.

The method of the present invention also relates to mixing a cationicsurfactant, a block copolymer, a population of polynucleotide moleculesand an amorphous cryoprotectant or a crystalline bulking agent and anycombination thereof at a temperature above the cloud point of said blockcopolymer. The cloud point is dependent upon the block copolymer used inthe mixture of the current invention. However, the suitable cloud pointcan range from about 1° C. to about 20° C. When CRL-1005 is the blockcopolymer, the temperature at which the mixture of the current inventionis mixed can range from about 8° C. to about 35° C.

To this end, the present invention also relates to a polynucleotidebased medicament formulation which first comprises a polynucleotide, anadjuvant component comprising a block copolymer, a cationic surfactantand an amorphous cryoprotectant or a crystalline bulking agent, asdescribed within this specification, and secondly comprising a non-ionicsurfactant, such as polysorbate-80 or other excipients, including butnot limited to excipients known in the art such as glycerol or propyleneglycol, or a non-ionic surfactant listed herein.

In a suitable embodiment of this invention, a formulation comprising apolynucleotide, a block copolymer, a cationic surfactant and anamorphous cryoprotectant or a crystalline bulking agent areco-solubilized at a temperature below the cloud point of the blockcopolymer. The presence of this cryoprotectant or bulking agent providesstability to the polynucleotide formulation during the freeze-dryingprocess and subsequent reconstitution. By “stability,” it is meant thataverage size and size distribution are not affected, i.e. that little orno fusion or aggregation is observed upon reconstitution.

Amorphous cryoprotectants which are suitable for use herein includeinter alia, mono, di, or oligosaccharides, polyols, and proteins such asalbumin; disaccharides such as sucrose and lactose; monosaccharides suchas fructose, galactose and glucose; poly alcohols such as glycerol andsorbitol; and hydrophilic polymers such as polyethylene glycol.

The amorphous cryoprotectant is suitably added to the formulations ofthe present invention before freezing, in which case it can also serveas a bulking agent. However, as a hydrophilic component, it may alsoprovide for enhanced liquid stability.

With regard to crystalline bulking agents, such agents are often used inthe preparation of pharmaceutical compositions to provide the necessarybulk upon lyophilization. Many types of crystalline bulking agents areknown in the art. (See, Martini et al. PDA J. Pharm Sci Tech51(2):62-61, 1997).

Exemplary crystalline bulking agents include D-mannitol, trehalose, anddextran. As the aforementioned are exemplary only, one skilled in theart would recognize that any compound which, when included in theformulations of the present invention during freezing or lyophilizationunder given conditions, forms crystals, would be considered a suitablecrystalline bulking agent. Within the context of the present invention acrystalline bulking agent is generally defined as a compound which canexist in a crystalline form and whose glass transition point (Tg) isbelow the temperature at which it is being freeze-dried. For example, aconventional freeze-dryer operates at a shelf-temperature from betweenabout −10° C. to about −50° C. Therefore, in one embodiment, acrystalline bulking agent has a Tg below about −50° C.

In a suitable embodiment, the solution comprises about 1% to about 20%(w/v) of the amorphous cryoprotectant or crystalline bulking agent. In asuitable embodiment, the solution contains about 3% to about 17%, about5% to about 15% or about 8% to about 12% (w/v) amorphous cryoprotectantor crystalline bulking agent. For example about 8%, about 9%, about 10%,about 11%, or about 12% (w/v) amorphous cryoprotectant or crystallinebulking agent.

Suitable for use in the present invention are cryoprotectants andbulking agents from the group consisting of, but not limited to thefollowing sugars: sucrose, lactose, trehalose, maltose or glucose. In asuitable embodiment, the solution comprises about 1% to about 20% (w/v)sugar. In a suitable embodiment, the solution contains about 3% to about17%, about 5% to about 15% or about 8% to about 12% (w/v) sugar. Forexample about 8%, about 9%, about 10%, about 11%, or about 12% (w/v)sugar.

In another suitable embodiment the solution contains about 1% to about20% (w/v) sucrose. In a suitable embodiment, the solution contains about3% to about 17%, about 5% to about 15% or about 8% to about 12% (w/v)sucrose. For example about 8%, about 9%, about 10%, about 11%, or about12% (w/v) sucrose. In yet another suitable embodiment, the solutioncontains about 10% (w/v) sucrose.

In a suitable embodiment of this invention, a formulation comprising apolynucleotide, a block copolymer, a cationic surfactant, an amorphouscryoprotectant or a crystalline bulking agent and a pH buffering agentare co-solubilized at a temperature below the cloud point of the blockcopolymer.

In another suitable embodiment, the solution comprising a blockcopolymer, cationic surfactant, polynucleic acid and amorphouscryoprotectant or crystalline bulking agent also contains a physiologicbuffer that maintains the solution pH within the range of about pH 4.0to about pH 9.0. In a suitable embodiment, the pH of the co-solubilizedmixture is about pH 5.0 to about pH 8.0, about pH 6.0 to about pH 8.0,or about pH 7.0 to about pH 7.5. For example, the pH of theco-solubilized mixture is about pH 7.0, about pH 7.1, about pH 7.2,about pH 7.3, about pH 7.4 or about pH 7.5.

pH buffering agents suitable for use in the invention include, but arenot limited to, saline, PBS,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),3-(N-Morpholino)propanesulfonic acid (MOPS),2-bis(2-Hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS),potassium phosphate (KP), sodium phosphate (NaP), dibasic sodiumphosphate (Na₂HPO₄), monobasic sodium phosphate (NaH2PO₄), monobasicsodium potassium phosphate (NaKHPO₄), magnesium phosphate(Mg₃(PO₄)₂—4H₂O), potassium acetate (CH₃COOK), D(+)-α-sodiumglycerophosphate (HOCH₂CH(OH)CH₂OPO₃Na₂) and other physiologic buffersknown to those skilled in the art. Additional pH buffering agents foruse in the invention include, a salt M-X dissolved in aqueous solution,association, or dissociation products thereof, where M is an alkalimetal (e.g., Li⁺, Na⁺, K⁺, Rb⁺), suitably sodium or potassium, and whereX is an anion selected from the group consisting of phosphate, acetate,bicarbonate, sulfate, pyruvate, and an organic monophosphate ester,preferably glucose 6-phosphate or DL-a-glycerol phosphate, and otherphysiologic buffers known to those skilled in the art. In a suitableembodiment of the invention, the pH buffering agent is selected from thegroup consisting of sodium phosphate, potassium phosphate, dibasicsodium phosphate (Na₂HPO₄), monobasic sodium phosphate (NaH₂PO₄),monobasic sodium potassium phosphate (NaKHPO₄), magnesium phosphate(Mg₃(PO₄)₂—4H₂O), potassium acetate (CH₃COOK), and D(+)-α-sodiumglycerophosphate (HOCH₂CH(OH)CH₂OPO₃Na₂).

In a suitable embodiment of the invention, the concentration of the pHbuffering agent is from about 5 mM to about 25 mM. Suitably, aformulation of the present invention may have a final pH buffering agentconcentration of about 7 mM to about 20 mM, or about 8 mM to about 15mM, or about 9 mM to about 12 mM. For example, a formulation of thepresent invention may have a final pH buffering agent concentration ofabout 9 mM, about 10 mM, about 11 mM, or about 12 mM. In anothersuitable embodiment, the concentration of the pH buffering agent isabout 10 mM.

In another suitable embodiment of the invention, the concentration ofthe pH buffering agent selected from sodium phosphate, potassiumphosphate, Na₂HPO₄, NaH₂PO₄, NaKHPO₄, Mg₃(PO₄)₂—4H₂O, andHOCH₂CH(OH)CH₂OPO₃Na₂ is from about 5 mM to about 25 mM. Suitably, aformulation of the present invention may have a final concentration ofpH buffering agent selected from sodium phosphate, potassium phosphate,Na₂HPO₄, NaH₂PO₄, NaKHPO₄, Mg₃(PO₄)₂—4H₂O, and HOCH₂CH(OH)CH₂OPO₃Na₂ ofabout 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM toabout 12 mM. For example, a formulation of the present invention mayhave a final concentration of pH buffering agent selected from sodiumphosphate, potassium phosphate, Na₂HPO₄, NaH₂PO₄, NaKHPO₄,Mg₃(PO₄)₂—4H₂O, and HOCH₂CH(OH)CH₂OPO₃Na₂ of about 9 mM, about 10 mM,about 11 mM, or about 12 mM. In another suitable embodiment, theconcentration of pH buffering agent selected from sodium phosphate,potassium phosphate, Na₂HPO₄, NaH₂PO₄, NaKHPO₄, Mg₃(PO₄)₂—4H₂O, andHOCH₂CH(OH)CH₂OPO₃Na₂ is about 10 mM. In an alternative embodiment, theconcentration of sodium phosphate is about 10 mM.

In one suitable embodiment of the invention, a formulation comprising apolynucleic acid, block copolymer, cationic surfactant, and an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,are co-solubilized at a temperature below the cloud point of the blockcopolymer. This mixture is then lyophilized.

In another suitable embodiment of the invention, a formulationcomprising a polynucleic acid, a block copolymer, cationic surfactant,an amorphous cryoprotectant or crystalline bulking agent, such as 10%(w/v) sucrose, and a suitable pH buffering agent, such as 10 mM sodiumphosphate, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This mixture is then lyophilized.

In another alternative embodiment, a formulation comprising apolynucleic acid, a block copolymer, cationic surfactant, and anamorphous cryoprotectant or crystalline bulking agent, such as 10% (w/v)sucrose, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This mixture is then sterilized via cold filteringprior to lyophilization.

In another suitable embodiment, a formulation comprising a polynucleicacid, a block copolymer, cationic surfactant, an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,and a suitable pH buffering agent, such as 10 mM sodium phosphate, areco-solubilized at a temperature below the cloud point of the blockcopolymer. This mixture is then sterilized via cold filtering prior tolyophilization.

The cold filtration step must take place at a temperature below thecloud point of the block copolymer comprised in the formulation. Thecold filtration step is suitably performed at a temperature betweenabout −2° C. to about 8° C. For example, the cold filtration step can beperformed at about −2° C., at about −1° C., at about 0° C., at about 1°C., at about 2° C., at about 3° C., at about 4° C., at about 5° C., atabout 6° C., at about 7° C. or at about 8° C.

The filtration of the cold solution (from about −2° C. to about 8° C.)of polynucleotide, block copolymer, and cationic surfactant provides acost-effective and time-efficient method by which to sterilize thesolution. This filtration step eliminates the need to pre-sterilize thepolynucleotide, block copolymer and cationic surfactant prior to mixing.By passing the mixture through a sterile filter with a defined pore sizesmaller than bacterial pathogens, the solution is sterilized. The poresize of the filters utilized in the cold filtration step in the presentinvention are suitably from about 0.01 microns to about 2 microns.Alternatively, the pore size of the filters utilized in the coldfiltration step in the present invention is about 0.05 microns to about0.25 microns. For example, pore size of the filters for the coldfiltration step can be about 0.05 microns, about 0.08 microns, about 0.1microns, about 0.15 microns, about 0.16 microns, about 0.17 microns,about 0.18 microns, about 0.19 microns, about 0.2 microns, about 0.21microns, about 0.22 microns, about 0.23 microns, about 0.24 microns, orabout 0.25 microns.

In an alternative embodiment of the invention, a formulation comprisinga polynucleic acid, block copolymer, cationic surfactant, and anamorphous cryoprotectant or crystalline bulking agent, such as 10% (w/v)sucrose, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This solution is then cycled through its cloudpoint temperature several times, prior to being sterilized via coldfiltering and subsequent freeze-drying.

In a suitable embodiment of the invention, a formulation comprising apolynucleic acid, block copolymer, cationic surfactant, an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,and a suitable pH buffering agent, such as 10 mM sodium phosphate, areco-solubilized at a temperature below the cloud point of the blockcopolymer. This solution is then cycled through its cloud pointtemperature several times, prior to being sterilized via cold filteringand subsequent freeze-drying.

In another suitable embodiment, a formulation comprising a polynucleicacid, the block copolymer CRL-1005, the cationic surfactant BAK, and anamorphous cryoprotectant or crystalline bulking agent, such as 10% (w/v)sucrose, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This solution is then lyophilized.

In another suitable embodiment, a formulation comprising a polynucleicacid, the block copolymer CRL-1005, the cationic surfactant BAK, anamorphous cryoprotectant or crystalline bulking agent, such as 10% (w/v)sucrose, and a suitable pH buffering agent, such as 10 mM sodiumphosphate, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This solution is then lyophilized.

In another alternative embodiment, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, the cationic surfactantBAK, and an amorphous cryoprotectant or crystalline bulking agent, suchas 10% (w/v) sucrose, are co-solubilized at a temperature below thecloud point of the block copolymer. This solution is then sterilized viacold filtering prior to lyophilization.

In another suitable embodiment, a formulation comprising a polynucleicacid, the block copolymer CRL-1005, the cationic surfactant BAK, anamorphous cryoprotectant or crystalline bulking agent, such as 10% (w/v)sucrose, and a suitable pH buffering agent, such as 10 mM sodiumphosphate, are co-solubilized at a temperature below the cloud point ofthe block copolymer. This solution is then sterilized via cold filteringprior to lyophilization.

In another embodiment of the invention, a formulation comprises apolynucleic acid, the block copolymer CRL-1005, the cationic surfactantBAK, and an amorphous cryoprotectant or crystalline bulking agent, suchas 10% (w/v) sucrose, are co-solubilized at a temperature below thecloud point of the block copolymer. This solution is then cycled throughits cloud point temperature several times, prior to being sterilized viacold filtering and subsequent freeze-drying.

In another suitable embodiment of the invention, a formulation comprisesa polynucleic acid, the block copolymer CRL-1005, the cationicsurfactant BAK, an amorphous cryoprotectant or crystalline bulkingagent, such as 10% (w/v) sucrose, and a suitable pH buffering agent,such as 10 mM sodium phosphate, are co-solubilized at a temperaturebelow the cloud point of the block copolymer. This solution is thencycled through its cloud point temperature several times, prior to beingsterilized via cold filtering and subsequent freeze-drying.

In another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, a cationic surfactantselected from the following group of cationic lipids:Bn-DHxRIE,DHxRIE-OAc, DHxRIE-OBz and Pr-DOctRIE-OAc, and an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,are co-solubilized at a temperature below the cloud point of the blockcopolymer. This solution is then lyophilized.

In another suitable embodiment of the invention, a formulationcomprising a polynucleic acid, the block copolymer CRL-1005, a cationicsurfactant selected from the following group of cationic lipids:Bn-DHxRIE, DHxRIE-OAc, DHxRIE-OBz and Pr-DOctRIE-OAc, an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,and a suitable pH buffering agent, such as 10 mM sodium phosphate, areco-solubilized at a temperature below the cloud point of the blockcopolymer. This solution is then lyophilized.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, a cationic surfactantselected from the following group of cationic lipids: Bn-DHxRIE,DHxRIE-OAc, DHxRIE-OBz and Pr-DOctRIE-OAc, and an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,are co-solubilized at a temperature below the cloud point of the blockcopolymer. This mixture is then sterilized via cold filtering prior tofreeze-drying.

In yet another suitable embodiment of the invention, a formulationcomprising a polynucleic acid, the block copolymer CRL-1005, a cationicsurfactant selected from the following group of cationic lipids:Bn-DHxRIE, DHxRIE-OAc, DhxRIE-OBz and Pr-DOctRIE-OAc, an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,and a suitable pH buffering agent, such as 10 mM sodium phosphate, areco-solubilized at a temperature below the cloud point of the blockcopolymer. This mixture is then sterilized via cold filtering prior tofreeze-drying.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, a cationic surfactantselected from the following group of cationic lipids: Bn-DHxRIE,DHxRIE-OAc, DHxRIE-OBz and Pr-DOctRIE-OAc, and an amorphouscryoprotectant or crystalline bulking agent, such as 10% (w/v) sucrose,are co-solubilized at a temperature below the cloud point of the blockcopolymer. This solution is then cycled through its cloud pointtemperature several times, prior to being sterilized via cold filteringand subsequent freeze-drying.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, a cationic surfactantselected from the following group of cationic lipids: Bn-DHxRIE,DHxRIE-OAc, DHxRIE-OBz and Pr-DOctRIE-OAc, an amorphous cryoprotectantor crystalline bulking agent, such as 10% (w/v) sucrose, and a suitablepH buffering agent, such as 10 mM sodium phosphate, are co-solubilizedat a temperature below the cloud point of the block copolymer. Thissolution is then cycled through its cloud point temperature severaltimes, prior to being sterilized via cold filtering and subsequentfreeze-drying.

In another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, the cationic lipidPr-DOctRIE-OAc, and an amorphous cryoprotectant or crystalline bulkingagent, such as 10% (w/v) sucrose, are co-solubilized at a temperaturebelow the cloud point of the block copolymer. This solution is thenlyophilized.

In another suitable embodiment of the invention, a formulationcomprising a polynucleic acid, the block copolymer CRL-1005, thecationic lipid Pr-DOctRIE-OAc, an amorphous cryoprotectant orcrystalline bulking agent, such as 10% (w/v) sucrose, and a suitable pHbuffering agent, such as 10 mM sodium phosphate, are co-solubilized at atemperature below the cloud point of the block copolymer. This solutionis then lyophilized.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, the cationic lipidPr-DOctRIE-OAc, and an amorphous cryoprotectant or crystalline bulkingagent, such as 10% (w/v) sucrose, are co-solubilized at a temperaturebelow the cloud point of the block copolymer. This mixture is thensterilized via cold filtering prior to freeze-drying.

In yet another suitable embodiment of the invention, a formulationcomprising a polynucleic acid, the block copolymer CRL-1005, thecationic lipid Pr-DOctRIE-OAc, an amorphous cryoprotectant orcrystalline bulking agent, such as 10% (w/v) sucrose, and a suitable pHbuffering agent, such as 10 mM sodium phosphate, are co-solubilized at atemperature below the cloud point of the block copolymer. This mixtureis then sterilized via cold filtering prior to freeze-drying.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, the cationic lipidPr-DOctRIE-OAc, and an amorphous cryoprotectant or crystalline bulkingagent, such as 10% (w/v) sucrose, are co-solubilized at a temperaturebelow the cloud point of the block copolymer. This solution is thencycled through its cloud point temperature several times, prior to beingsterilized via cold filtering and subsequent freeze-drying.

In yet another embodiment of the invention, a formulation comprising apolynucleic acid, the block copolymer CRL-1005, the cationic lipidPr-DOctRIE-OAc, an amorphous cryoprotectant or crystalline bulkingagent, such as 10% (w/v) sucrose, and a suitable pH buffering agent,such as 10 mM sodium phosphate, are co-solubilized at a temperaturebelow the cloud point of the block copolymer. This solution is thencycled through its cloud point temperature several times, prior to beingsterilized via cold filtering and subsequent freeze-drying.

These example and equivalents thereof will become more apparent to thoseskilled in the art in light of the present disclosure and theaccompanying claims. It should be understood, however, that the examplesare designed for the purpose of illustration only and not limiting ofthe scope of the invention in any way. All patents and publicationscited herein are fully incorporated by reference herein in theirentirety.

EXAMPLES Example 1

Aim: Prepare a DNA/poloxamer/BAK formulation (5 mg/ml DNA, 7.5 mg/mLCRL-1005, 0.3 mM BAK) in 10% sucrose, 10 mM sodium phosphate vehicle andlyophilize the formulation and determine the effect on particle size ofthe process.

Apparatus: A 15 ml round bottom flask, with a ⅜″× 3/16″ egg-shapedmagnetic stirrer bar (Bel-art products) and a coming stirrer/hotplateand an ice bath.

Method: The required volume of DNA (6.1 mg/mL DNA, VR 4700, in 10%sucrose, 10 mM Sodium phosphate), was placed into the 15 mL round bottomflask and the solution stirred with a magnetic stirrer bar, in an icebath on top of a Corning stirrer/hotplate (speed 4, hotplate off) for 10minutes. The CRL-1005 was then added using a positive displacementpipette and the solution stirred for a further 30 minutes on ice. Therequired volume of BAK solution to give a final concentration of 0.3 mMwas then added drop wise, slowly, to the stirring solution over 1 minuteusing a iml pipette. The solution at this point was clear since it wasbelow the cloud point of the poloxamer and was stirred on ice for 30minutes. The ice bath was then removed and the solution stirred atambient temperature for 15 minutes to produce a cloudy solution as thepoloxamer passed through the cloud point.

The flask was then placed back into the ice bath and stirred for afurther 15 minutes to produce a clear solution as the mixture cooledbelow the cloud point of the CRL-1005. The ice bath was again removedand the solution stirred for a further 15 minutes. Stirring for 15minutes above and below the cloud point (total of 30 minutes), wasdefined as one thermal cycle. The mixture was cycled two more times. Thesolution was then diluted 1:2 with PBS and a 20 μL aliquot of thesolution was then removed, diluted in 2 mL of filtered (0.2 μm) PBS andthe particle size determined using a Malvern 3000 HS Zetasizer.

The formulation was then filtered sterilized. A 50 mL Steriflipfiltration system was placed in an ice bucket, with a vacuum lineattached and left for 1 hour to allow the device to equilibrate to thetemperature of the ice. The poloxamer formulation was then filteredunder vacuum, below the cloud point and then allowed to warm above thecloud point. A 20 μL aliquot of the solution was then removed, dilutedin 2 mL of filtered (0.2 μm) PBS and the particle size determined.

Three 5 mL borosilicate vials (Wheaton) were then filled with 1 mL eachof the formulation and the vials placed in a computer controlled VirtisAdvantage freeze dryer. Initially the vials were cooled below −40° C.for at least two hours and then the condenser was cooled to below −40°C. and the vacuum reduced to below 300 mTorr. The first step in primarydrying was to hold the vials at −40° C. for one hour, under a vacuum of120 mTorr. Then the temperature was raised to 20° C. over eight hoursand the vacuum maintained at 120 mTorr. After eight hours thetemperature and vacuum were maintained for a further one-hour. Thesecondary drying step involved raising the temperature to 30° C. over 30minutes and holding this temperature for a further two hours, whilemaintaining a vacuum of 120 mTorr. Finally the temperature was reducedto 20° C. over 30 minutes; the vials were sealed with grey butyl rubberstoppers (WestDireet) under vacuum and the samples removed for analysis.

A similar procedure was used to prepare microparticles forlyophilization in PBS. The formulation contained final concentrations of5 mg/ml DNA, 7.5 mg/mL CRL-1005, 0.3 mM BAK. The lyophilized particlesin PBS were compared to the lyophilized particles in the 10% sucrose, 10mM Sodium phosphate solution. Z average mean particle size andpolydispersity were measured using a Malvern 3000 HS Zetasizer for bothtype of particles before and after lyophilization and the result areshown in Table 1.

TABLE 1 Z average Vehicle mean (nm) Polydispersity Before PBS 247.2 0.04Lyophilization After PBS 268.8 0.59 Lyophilization Before 10% Sucrose223.9 0.07 Lyophilization 10 mM NaP After 10% o Sucrose 205.4 0.10Lyophilization 10 mM NaP

Example 2

One of the lyophilized samples as prepared in the 10% sucrose, 10 mMSodium phosphate solution as in Example 1 was reconstituted with 960 μlof sterile water for injection and gently mixed by hand and left on thebench top for 15 minutes. A 20 μl aliquot of this solution was thenremoved at 15, 60, 120, 240, 360 minute intervals, diluted in 2 ml offiltered (0.2 μm) 10% sucrose, 10 mM Sodium phosphate. The Z averagemean and polydispersity of the particles in these aliquots were measuredusing a Malvern 3000 HS Zetasizer as above (FIGS. 1A and IB).

Example 3

Sterile formulations containing DNA, CRL-1005 and other poloxamers withcationic ionic lipids, including but not limited to, benzalkoniumchloride in 8.5% sucrose can be prepared as described herein. Themixture is then lyophilized, and when reconstituted, these formulationsmay be used in immunogenicity studies. The T-cell responses of animalsinjected with the formulations described above can be measured by IFN-γELISpot assay and antigen-specific antibodies can be measured by ELISA.From the data, biologically active formulations with advantageousphysical or pharmaceutical properties and/or formulations with enhancedbiological activity can then be identified.

Immunogenicity studies can be conducted using the experimental protocolas described below. Groups of nine, six- to eight-week old BALB/c mice(Harlan-Sprague-Dawley) will receive bilateral (50 μL/leg) intramuscular(rectus femoris) injections of naked plasmid DNA or formulated plasmidDNA. The plasmid (VR4700) to be injected in all mice encodes theinfluenza nucleoprotein (NP). All mice will be boosted on(approximately) days 21 and 49. Sera will be collected fromNP-vaccinated mice after the third (˜ day 60) vaccination, andNP-specific antibody responses will be measured by ELISA. Two weeksafter the last immunization, splenocytes will be harvested from threemice/group/day for three sequential days, and antigen specific T-cellresponses will be measured by IFN-γ ELISpot assay.

The NP-specific antibodies produced in response to DNA vaccination willbe evaluated by ELISA. Briefly, 96 well Costar hi-binding ½ well ELISAplates are coated with 2 μg/mL of recombinant NP protein (Imgenex, SanDiego, Calif.) and blocked with 10% fetal bovine serum (FBS) in PBS.Wells are incubated with serial dilutions of each immune serum, andbound anti-NP antibody is detected by the sequential addition ofalkaline phosphatase-labeled goat anti-mouse IgG-Fcy and thecolorimetric substrate, p-nitrophenylphosphate. Conversion of thesubstrate is quantified at 405 nm.

The end-point dilution titer is defined as the reciprocal dilution atwhich the optical density at 405 run is greater than twice that measuredin wells containing assay buffer alone (i.e., the background value). Anaverage absorbance of eight wells containing assay buffer is used toestablish the background value. Wells incubated with a pool of sera fromNP DNA-vaccinated mice serve as a positive control.

T-cell responses to the DNA vaccines will be determined by quantifyingthe number of splenocytes secreting IFN-γ in response toantigen-specific stimulation as measured by IFN-γ ELISpot assay.Splenocyte cultures will be grown in RPMI-1640 medium containing 25 mMHEPES buffer and L-glutamine and supplemented with 10% (v/v) FBS, 55 μMβ-mercaptoethanol, 100 U/mL of penicillin G sodium salt, and 100 μg/mLof streptomycin sulfate. ImmunoSpot plates (Cellular Technology Limited,Cleveland, Ohio) are coated with rat anti-mouse IFN-γ monoclonalantibody (BD Biosciences, San Diego, Calif.), and blocked with RPMI-1640medium. Splenocyte suspensions can be produced from individualvaccinated mice and seeded in ELISpot plates at 1×10⁶, 3×10⁵, or 1×10⁵cells/well in RPMI medium containing 1 μg/mL of the appropriate MHCclass I-restricted peptide (M84, ²⁹⁷AYAGLFTPL³⁰⁵, Imgenex, San Diego,Calif.; NP, ¹⁴⁷TYQRTRALV ¹⁵⁵, Sigma/Genosys, The Woods, Tex.) or 20μg/mL of protein antigen with (CD8+ T cell ELISpot assay) or without(CD4+ T cell ELISpot assay) 1 U/mL of recombinant murine IL-2 (Roche,Indianapolis, Ind.). Control wells contain 1×10⁶ splenocytes incubatedin medium with or without IL-2 only (no antigen). After a 20-hourincubation at 37° C., captured IFN-γ is detected by the sequentialaddition of biotin-labeled rat anti-mouse IFN-γ monoclonal antibody andavidin-horseradish peroxidase. Spots produced by the conversion of thecolorimetric substrate, 3-amino-9-ethylcarbazole (AEC), are quantifiedby an ImmunoSpot reader (Cellular Technology Limited, Cleveland, Ohio).

1. A method of preparing a lyophilized composition comprising: (a)mixing (i) a polyoxyethylene (POE) and polyoxypropylene (POP) blockcopolymer; (ii) a polynucleotide; (iii) a cationic surfactant; and (iv)an amorphous cryoprotectant or a crystalline bulking agent; at atemperature below the cloud point of said block copolymer to form amixture; and (b) lyophilizing the mixture.
 2. The method of claim 1,further comprising a cold filtration step.
 3. The method of claim 1,wherein said mixture additionally comprises a pH stabilizing physiologicbuffer.
 4. A product produced by the process of claim
 1. 5. A stable,mono-dispersed product produced by reconstituting the product of claim 4with an aqueous solution.
 6. A product produced by the process of claim2.
 7. A stable, mono-dispersed product produced by reconstituting theproduct of claim 6 with an aqueous solution.
 8. A product produced bythe process of claim
 3. 9. A stable, mono-dispersed product produced byreconstituting the product of claim 8 with an aqueous solution.